I am going to compare the protein expression between two groups of mice by doing immunohistochemistry on tissue sections and subsequently confocal microscopy. The protein is expressed in the cell body of a specific cell type, and I want to see if it is up- or downregulated in the disease group. What is the best way to go about doing this, without introducing bias? I can't really do random selection of tissue sections, because there may be a gradient in expression along the mediolateral/dorsoventral extent. Would an option be to take several images per section and then average the intensity?
Any help would be appreciated!
Hello,
Since these are different biological samples, you must treat the samples exactly the same way for staining --- it will never be perfect, but hopefully with some planning you will be able to reduce variability (fixation times, washes, etc.). Make sure you use the same blend of antibody for each sample (if you are using NeuN and xxxxx antibody, mix these together in the same lot and split it between the two samples... do the same with the secondaries). For imaging, use the WT animal as a baseline for your protein of interest and reuse the exact same settings for your diseased animal sample. Use a tile scan on these sections (the same regions in each sample) in case there is a gradient. What kind of tissue samples are you using? I hope this helps!
Hello Elliot,
Thank you! I am using tissue sections from brain, they are 40 um thick. How would you suggest to compare the fluorescence intensities between the two groups from a tile scan? I also realise that, for instance, the number of positive cells within a frame would affect the intensity. Do you know how to avoid this kind of bias?
Again, thanks for your input!
Nina
This all depends on what kind of program you are using to capture the image. Certain software packages contain quantification tools (check with your confocal user manual). Often times, differences will be obvious just from looking at the image. If you want to quantify, I would assess individual cells that are positive for you stain (perhaps pair your staining with DAPI to differentiate single cells) and quantify fluorescence intensity (through your confocal analysis software or ImageJ for example). Use the Maximum intensity projection for the images (all of the slices from the stack put together) when analyzing. Make sure you are using the same bregma for your brain slices to ensure continuity between samples. I personally have not done much quantification--- perhaps other experts have more direction in that area. Let me know if you need any more clarification!
All the best,
E
CTCF may be an option for analysis, see: https://sciencetechblog.files.wordpress.com/2011/05/measuring-cell-fluorescence-using-imagej.pdf
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